Effects of Different Storage Media, Temperature, and Time on Osteoblast Preservation in Autogenous Bone Grafts: A Histomorphometrical Analysis

Statement of the Problem: Autogenous bone graft is the gold standard for bone reconstruction. Osteogenic cells must be kept viable in graft for a successful procedure. In extracorporeal preservation of grafts during surgery, three different factors may influence the quality of grafts. These factors include temperature, storage medium, and time interval. Purpose: In this study, we evaluated the effects of different storage media, temperatures, and times on osteoblast count in autogenous bone grafts, preserved extracorporeally. Materials and Method: Samples were obtained from iliac crest region in a goat. The grafts were preserved in 36 groups of different storage time, temperature, and medium. Samples were histomorphometrically analyzed to determine osteoblast count as the criteria of graft quality. Results: In almost all samples, room temperature was the most and incubator was the least favorable storage temperatures. In grafts preserved in room temperature, no difference was noted between normal saline and Ringer’s lactate solution and in almost all of the samples autologous blood and dry environment were more favorable media than Ringer’s lactate solution. The effect of storage time was highly depended on the combination of temperature and solution. Conclusion: The results demonstrated that for preserving as many osteoblasts as possible in bone grafts, the best temperature was room temperature and the least favorable temperature was incubator. In addition, when bone fragments were preserved in room temperature, the best medium for graft storage was blood, which showed better results than normal saline and Ringer’s lactate solution.

Normal saline solution and Ringer's lactate solution are the most common infusion fluids in operation room settings. Normal saline solution is also the most common solution used in surgeries as coolant and irrigation [4]. Most surgeons also consider whole blood a proper medium for bone graft maintenance and it can easily be obtained during surgery [5]. Although air exposure is considered the most inappropriate medium for bone graft maintenance, in clinical practice the grafts are often stored in dry environments [7].
Rocha et al. [8] studied the effects of preserving bone grafts for 30 minutes in different storage media including normal saline solution, PPP, and dry environment in comparison with a control group, which was implanted immediately. The results showed more empty lacunae in the dry group when compared with the control group, the saline group, or the PPP group. No significant difference was reported in the count of empty lacunae between the saline group, the PPP group, and the control group.
Another factor is preservation temperature. There are three temperature ranges usually possible in operation rooms, during surgeries. These include cold preservation (2-8˚ᶜ), room temperature (18-24˚ᶜ), and incubator (37˚ᶜ). Antonenas et al. [9] preserved blood stem cells in room temperature and refrigerator for 24 hours, 48 hours, and 72 hours. His study showed a great loss of viable cells in the grafts stored in room temperature (21.9%) compared to those stored in refrigerator (9.4%).
Finally, the time interlude that a graft is preserved extracorporeally, can affect the quality of the bone graft.
Williams et al. [10] stored canine femoral condyles at 4˚ᶜ for 14, 21, and 28 days. His study showed >95% cell viability at 14 days, 75-98% at 21 days, and 65-90% at 28 days of preservation. In this study, we have assessed the effects of three major factors on regenerative potential of autogenous cancellous bone grafts.

Material and Method
A one-year-old healthy brown female goat (Capraaegagrus hircus) weighing 28 kg was chosen as the animal model. This study was done according to International animal rights with adherence to the animal experiment rules assigned by Shiraz University of Medical Scienc-es. Before the surgery, 30ᶜᶜ of blood was obtained from right jugular vein under aseptic condition. The blood was mixed with 6ᶜᶜ of sodium citrate 3.8%, as anticoagulant. The blood was transferred to previously sterilized and labeled laboratory tubes assigned for blood as storage medium. Other tubes contained sterile normal saline solution; Ringer's lactate solution, or they were empty.  The counting frame had two borders of inclusion and two borders of exclusion. The initial field was selected randomly out of the sample section; the remaining fields were selected by moving the microscope stage using microscope stage along X-and Y-axis in equal intervals. An oil immersion lens with ×100 magnification was used. To assess the numerical density of osteoblasts, the focus area was also moved on Z-axis.
Through traveling on Z-axis and using magnification of 60×, a microcater (Hidenhain MT-12, Germany) which calculated the Z-axis movements was employed.
Each plane thickness is 5µm, but the first nuclei that came into focus, were excluded. Within the next 5µm of traveling on Z-axis (height) any nucleolus which came into maximal focus was counted, if located within the counting frame or in contact with the inclusion border and did not touch the exclusion border or the frame.
Numerical density of osteoblasts was calculated by the following formula [11]: N v = In this equation, ƩQ represents the sum of the counted osteoblasts for each sample, a(f) represents the frame area and Ʃp represents the total number of fields in Xand Y-axis on which osteoblasts were counted.
Kruskal-Wallis non-parametric test was used for data analysis and a p Value <0.05 was considered statistically significant. SPSS statistical software (version 15) was utilized for statistical analysis.

Storage Media Comparison
Four groups of storage media were compared with each other sorted and layered by similar time interval and storage temperature, each group contained five specimens. Table 1 shows different storage media comparisons in different preservation temperatures and times.
1. Comparison of four storage media groups, which preserved bone grafts for 2 hours in cold temperature, showed that the highest count was noted in samples stored in autologous blood, followed by dry environment, Ringer's lactate solution, and finally normal saline solution respectively (p< 0.05).

Comparison of four storage media groups which
preserved bone grafts for 4 hours in cold temperature showed the highest count was noted in samples stored in normal saline solution, followed by autologous blood, dry environment and finally Ringer's lactate solution respectively (p<0.05).
3. Comparison of four storage media groups which preserved bone grafts for 12 hours in cold temperature showed that the highest count was noted in samples stored in dry environment, followed by Ringer's lactate solution, normal saline solution, and autologous blood, respectively (p<0.05).

Comparison of four storage media groups, which
preserved bone grafts for 2 hours in room temperature, showed the highest count was noted in samples stored in autologous blood, followed by ringer lactate or normal saline solutions and finally dry envir-

Storage Time Comparison
Three groups of storage time were compared with each other sorted and layered by similar storage temperature and medium, each group contained five specimens. Table 3 shows different storage times comparison in different preservation media and temperatures. Antonenas et al. [9] showed more viable cells in grafts stored in normal saline solution in cold preservation than those kept in room temperature for 24, 48 or 72 hours; in this study, the specimens stored in normal saline for 4 or 12 hours, showed higher count of osteoblast in cold preservation than room temperature.
In comparing different time-periods the grafts were kept, in those preserved in room temperature, the grafts that were stored for 12 hours showed the least count of osteoblasts, except for the specimens stored in dry environment. In all bone grafts stored in autologous blood, those preserved for 12 hours showed less count of osteoblasts than those kept for 2 or 4 hours. However, the graft particles, which were stored for 12 hours in dry environment, showed higher count of osteoblast in comparison with those, preserved for 2 or 4 hours.